Larrea tridentata, also known as Larrea divaricata, Larrea, chaparral, or creosote bush, is a shrubby plant which dominates some areas of the desert southwest in the United States and Northern Mexico as well as some desert areas of Argentina. Tea made from the leaves of Larrea tridentata has long been used in folk medicine to treat digestive disorders, rheumatism, venereal disease, sores, bronchitis, chicken pox, and the common cold.
According to Masayuki Sakakibara, et al., Flavonoid Methyl Ethers on the External Leaf Surface of Larrea Tridentata and L. Divaricata in PHYTOCHEMISTRY, vol. 15, pp. 727-731 (Pergamon Press 1976), the disclosure of which is incorporated herein by reference, the natural products on the surface of the Larrea tridentata leaves, the leaf resin, constitutes approximately 10-15% of the dry weight of the leaves and is composed of approximately 50% nordihydroguaiaretic acid ("NDGA") and related lignans, and 50% flavonoids. NDGA, extracted from Larrea tridentata by an alkaline extraction method (U.S. Pat. No. 2,382,475, incorporated herein by reference) and produced synthetically (U.S. Pat. No. 2,644,822, incorporated herein by reference) was used as an antioxidant in edible fats, butter, oils and oleaginous materials (U.S. Pat. No. 2,373,192, incorporated herein by reference), until the GRAS (Generally Recognized As Safe) status of NDGA was revoked after animal studies revealed evidence of kidney toxicity resulting from the ingestion of NDGA.
NDGA is known as a powerful antioxidant compound. However, NDGA can itself be oxidized to toxic oxidation products by chemical means or by oxidation during processing and storage. A highly reactive and toxic oxidation product of NDGA is nordihydroguaiaretic acid ortho di-a-b-unsaturated quinone ("NDGA quinone"), which according to T. J. Mabry et al., The Natural Products Chemistry of Larrea in CREOSOTE BUSH: BIOLOGY AND CHEMISTRY OF LARREA IN THE NEW WORLD DESERTS, ch. 5, pp. 115-133 (Dowden, Hutchinson and Ross, Pennsylvania 1977) (incorporated herein by reference), occurs in Larrea and Larrea extracts and probably serves as a toxin to protect the plant from being eaten by herbivores. According to a recent report by FDA scientists (W. R. Obermeyer et al., Chemical Studies of Phytoestrogens and Related Compounds in Dietary Supplements: Flax and Chaparral, 208 PROC. SOC. EXP. BIOL. MED., pp. 6-12 (1995), the disclosure of which is incorporated herein by reference), NDGA quinone is found in chaparral (Larrea tridentata) and is suspected to be a causative agent of the toxic effects associated with consumption of chaparral products.
NDGA is the dominant lignan present in Larrea tridentata NDGA is known to possess a variety of biological effects including anti-tumor activity, enzyme inhibition activity and antimicrobial activity according to W. Donald MacRae & G. H. Neil Towers, Biological Activities of Lignans, PHYTOCHEMISTRY, vol. 23, pp. 1207-1220 (Pergamon Press 1984) (incorporated herein by reference). Additionally, NDGA and other antioxidants have been shown to be potent inhibitors of the human immunodeficiency virus type 1 (HIV) transcription. The mode of action of this anti-HIV activity was suggested to be due to the potent antioxidant activity of NDGA inhibiting a redox regulated signal transduction pathway leading to production of HIV virus.
More recently, three scientific articles, John N. Gnabre et al., Inhibition of human immunodeficiency virus type 1 transcription and replication by DNA sequence-selective plant lignans, PROC. NATL. ACAD. SCI., USA, vol. 92, pp. 11239-11243 (November 1995); John Gnabre et al., Characterization of Anti-HIV Lignans from Larrea tridentata, TETRAHEDRON, vol. 51, pp. 12203-12210 (1995); and John Noel Gnabre et al., Isolation of anti-HIV-1 lignans from Larrea tridentata by counter-current chromatography, JOURNAL OF CHROMATOGRAPHY A, vol. 719, pp. 353-364 (1996), the disclosure of all three articles being incorporated herein by reference, demonstrate that at least two lignans isolated from Larrea tridentata, NDGA and 3-O-methyl nordihydroguaiaretic acid ("Mal. 4") inhibit transcription and replication of human immunodeficiency virus type 1 by a novel mechanism. This elucidated mechanism of anti-HIV activity is thought to be due to the ability of the two identified Larrea tridentata lignans, NDGA and Mal. 4, to interfere with the binding of the Sp1 protein to Sp1 binding sites in the HIV long terminal repeat (HIV-LTR). According to this theory, by inhibiting Sp1 binding in the HIV-LTR, the promoter activity of the HIV-LTR is eliminated so that HIV transcription, HIV Tat-regulated transactivation and HIV replication do not occur. It is further theorized by the authors that viruses other than HIV, which require binding of Sp1 protein in promoter-containing Sp1 binding sites to initiate viral replication, might also be inhibited by the anti-HIV lignans isolated from Larrea tridentata and that this class of lignans, in general, may possess a broader antiviral action of important interest.
Also, in a recent article, Anneke K. Raney & Alan McLachlan, Characterization of the Hepatitis B Virus Large Surface Antigen Promoter Sp1 Binding Site, VIROLOGY, vol. 208, pp. 399-404(1995), binding sites for the transcription factor Sp1 have been identified in the DNA promoter regions of at least two important viral genes of the Hepatitis B virus (HBV) which may be involved in the coordinate regulation of HBV transcription by transcription factor Sp1.
Kaposi's Sarcoma, a cancer that frequently occurs among AIDS patients, has recently been implicated to be caused by a new herpes virus, human herpes virus-8 (HHV-8). See Roland G. Nador et al., Primary Effusion Lymphoma: A Distinct Clinicopathologic Entity Associated With the Kaposi's Sarcoma--Associated Herpes Virus, BLOOD, vol. 88, no. 2, pp. 645-656 (Jul. 15, 1996), incorporated herein by reference. See also, Matt Crenson, Kaposi's Sarcoma is tied to herpes, THE PHILADELPHIA INQUIRER, p. A4 (Jul. 31, 1996), and Lawrence K. Altman, Aids Cancer Said to Have Viral Source: Breakthrough Seen in Kaposi's Sarcoma, NEW YORK TIMES .sctn. A, p. 22 (Feb. 1, 1995), incorporated herein by reference.
Certain flavonoid compounds, especially members of the chemical classes flavones and favonols can inhibit HIV activation at fairly low concentrations (See, J. William Critchfield et al., Inhibition of HIV Activation in Latently Infected Cells by Flavonoid Compounds" in AIDS Research and Human Retroviruses, AIDS RESEARCH AND HUMAN RETROVIRUSES, vol. 12, no. 1, pp. 39-46 (1996), incorporated herein by reference). As further cited in Masayuki Sakakibara, et al., Flavonoid Methyl Ethers on the External Leaf Surface of Larrea Tridentata and L. Divaricata in PHYTOCHEMISTRY, vol. 15, pp. 727-731 (Pergamon Press 1976), Larrea tridentata contains an abundance of these classes of antiviral flavonoids, particularly methyl ethers of flavonols.
Like many physiologically active chemicals isolated from plant sources, these antiviral lignans and flavonoid compounds appear to work synergistically with other unresolved compounds present in crude extracts of Larrea tridentata. The identity and mode of action of these synergistic compounds is unknown but they may facilitate absorption of the antiviral lignans or otherwise enhance the specific physiological, antiviral effects.
NDGA is known to be a potent inhibitor of the enzyme 5-lipoxygenase. One of the enzymatic products of 5-lipoxygenase is 5-hydroperoxyeicosatetraenoic acid (HPETE) which is the precursor compound for the biosynthesis of very potent chemical mediators of inflammation, known as leukotrienes. As detailed in William R. Henderson, The Role of Leukotrienes in Inflammation, ANN. INTER. MED., vol. 121, pp. 684-697 (1994), the disclosure of which is incorporated herein by reference, 5-lipoxygenase is limited to a specific number of myeloid cells including: neutrophils, eosinophils, monocytes, macrophages, mast cells, basophils and B-lymphocytes. Leukotrienes are chemicals which induce prolonged muscle contraction, especially in the bronchioles of the lungs, and also increase vascular permeability and attract neutrophils and eosinophils to the site of inflammation. The leukotrienes play a major role in the inflammatory response to injury. Leukotrienes have also been implicated in the pathogenesis of several inflammatory diseases including: asthma, psoriasis, rheumatoid arthritis and inflammatory bowel disease. The role of leukotrienes as mediators of inflammatory diseases makes them attractive targets for therapeutic drugs to treat these diseases.
Many inhibitors of leukotriene synthesis are being developed. Recently, a 5-lipoxygenase inhibitor, Zileuton, was found to be effective in the treatment of asthma during clinical tests (Elliot Israel et al., Effect of Treatment With Zileuton, a 5-Lipoxygenase Inhibitor, in Patients With Asthma, JAMA, vol. 275, pp. 931-936 (Mar. 27, 1996), the disclosure of which is incorporated herein by reference). The success of Zileuton underscores the utility and need of therapeutic agents containing 5-lipoxygenase inhibitors in the treatment of inflammatory disease processes, including asthma.
Throughout this specification and claims, viral diseases are intended to include all diseases, attributed to a pathological virus of humans or animals, in which the causative viral agent which requires the Sp1 class of proteins to initiate viral replication, including certain viral agents of venereal diseases such as the Herpes viruses (the Herpesviridae), HSV-1 and HSV-2, viral hepatitis (the Hepadnaviridae) such as hepatitis B, and members of the retrovirus family (the retroviridae) including Varicella-Zoster viruses, cytomegalovirus (CMV), the human T-lymphotrophic viruses 1 and 2 (HTLV-1 and (HTLV-2) the human immunodeficiency viruses 1 and 2 (HIV-1 and HIV-2) and the cancer Kaposi's Sarcoma. Inflammatory diseases, throughout the specification and claims, are intended to include all diseases in which leukotrienes are known to play a major role or have been implicated including: asthma, allergic rhinitis, psoriasis, rheumatoid arthritis, inflammatory bowel disease, inflammatory pain, cystic fibrosis, adult respiratory distress syndrome, glomerulonephritis, inflammation of the skin, and virally induced inflammation (caused by CMV and other members of the Herpesviridae) leading to atherosclerosis/arteriosclerosis and subsequent coronary artery disease.
In light of the foregoing background, there exists the need for a commercial method of producing a Larrea tridentata extract which contains a high concentration of both the identified antiviral lignans (NDGA and Mal. 4), flavonoids, and a wide variety of other associated organic compounds from the leaf resin, which may contribute synergistic antiviral and lipoxygenase inhibitory activity.
Additionally, for the purpose of toxicological safety, it is of critical importance that the Larrea tridentata extract, to be used for medical applications, be processed to reduce the concentration of the toxic compound, NDGA quinone, which is reported to occur naturally in Larrea tridentata plant tissues. There is also a need to inhibit the natural production of toxic oxidation products, such as NDGA quinone, in the Larrea tridentata extract during processing and storage of the extract and formulated products and to facilitate the processing of the concentrated extract as either a liquid, slurry, or solid.
Lastly, there is a need for products comprising synthesized NDGA which is stabilized against oxidation into NDGA quinone during processing and storage.